Engrailed (En) is a transcription factor first discovered in Drosophila but later found to be present in all animals, playing an important role in controllin neuronal development. Little is known about the cell surface molecules that it regulates. The long-term goal of this research is to find out how En regulates synaptic connectivity in the CNS, with a particular focus on identifying and characterizing its target genes of cell surface effector molecules. Preliminary data show that overexpression of En in Drosophila olfactory neurons alters their axonal path finding to their targets, the olfactory glomeruli. Additionally, ectopic E expression in a normally En- negative subset of auditory neurons allows them to form synaptic connections with the Giant Fiber (GF) escape neuron. The first aim is to selectively knock out En in olfactory neurons using fly lines in which RNAi is driven by the Gal4-UAS system. I will determine the effects on axonal guidance by assaying changes in the morphology of GFP-labeled olfactory axons, and alterations in the positions of immunolabeled olfactory glomeruli. The second aim will drive En RNAi in the auditory neurons and measuring their synaptic input to the GF. The third aim is to test whether increasing or knocking down expression of the En target Connectin, a member of the conserved LRR superfamily of adhesion molecules, alters axon guidance or glomerulus positioning. I will also test whether En knockout results in overexpression of this protein, as would be expected if it is a target of En repression. In the finl aim, I will ectopically express or knock down the En-binding target gene Neuroglian, a cell surface adhesion molecule homologous to vertebrate L1-CAM, then use the auditory synapse assays of connection to the GF. I will also test whether its immunostaining is altered by overexpression or knockout of En, as would be expected if it is negatively regulated by En. Relevance: En has been shown to control the survival of midbrain dopaminergic neurons, with En knockout mice showing Parkinson-like symptoms, and it has also been linked to autism spectrum disorder. Drosophila models are particularly useful for the discovery of molecular pathways that are directly relevant to human health, because most of these pathways have been conserved during evolution. All animals have En protein, so it is very likely that any molecules that are regulated by it during the process of synapse formation in Drosophila have their counterparts in humans, playing similar roles. These molecules may be of great potential importance in neurological diseases such as Parkinson's or autism. In addition, the basic knowledge gained from this project about the development of dipteran olfactory and auditory systems could be of use in designing ways to disrupt the feeding and mating patterns of mosquitoes, which are vectors of diseases such as malaria and dengue fever. PUBLIC HEALTH RELEVANCE: This research will help our understanding of the molecular pathways involved in the formation of synaptic connections in the central nervous system, using the fruit fly, Drosophila melanogaster, as a model system. The long-term goal of this research is to find out how a protein that is present in all animals' brains regulates the accuracy of the formation of synaptic connections, by controlling the presence of other cell surface molecules. The results of this study may have relevance to diseases of humans such as Parkinson's disease and autism, and may help in the control of disease-transmitting insects such as mosquitoes.